Keratin-based particles for protection and restoration of hair properties.

OBJECTIVE: Human hair is an element with unquestionable relevance in society both for women and men. Therefore, it is of great importance to develop new cosmetic products for hair care capable to restore and improve hair's characteristics. Here, we explore the potential of keratin-based particles in the protection and recovery of hair mechanical properties and thermal stability. METHODS: Keratin-based particles were obtained by high pressure homogenization (HPH) using keratin and silk fibroin. The particles were characterized regarding size, superficial charge and polydispersity index. Their safety to cells was assessed using human skin keratinocytes. Virgin and overbleached Asian hair were treated with eight keratin-based formulations. The effect of particles on hair's mechanical properties was evaluated in terms of stiffness and tensile strength. The impact of treatments in hair thermal performance was studied using differential scanning calorimetry (DSC). RESULTS: Keratin-based particles were capable to recover and/or improve the mechanical properties of virgin and overbleached hair. Virgin hair treated with K80 SF20 P particles presented an improvement in the mechanical properties of around 40%. An increase in keratin α-helix denaturation enthalpy and in surface smoothness for both types of hair was also verified after treatment. These particles demonstrated stability over time and proved to be safe when tested in human keratinocytes. CONCLUSION: The keratin-based particles here presented have the potential to be incorporated in the development of new and effective hair care cosmetic formulations.

Effect of a peptide in cosmetic formulations for hair volume control.

OBJECTIVE: The capacity of hair to absorb water causes changes in its physical and cosmetic properties under different environmental conditions. Hence, the control of hair volume in variable relative humidity settings is an important topic in cosmetics. The behaviour of two types of hair, Caucasian and Asian, was studied regarding their volume change in different relative humidity conditions. The ability of a peptide as a hair volume treatment was evaluated in two climate control formulations. METHODS: Tresses of the two types of hair were tested in two relative humidity (RH) conditions: (A) variable relative humidity (2 h 40% RH, followed by 2 h 90% RH and 2 h of 40% RH), and (B) continuous high relative humidity (90% RH for 6 h). Changes in the hair tress volume were assessed throughout time. Hair treated with two climate control formulations, with and without a peptide (KP peptide), were tested under the two relative humidity conditions. RESULTS: Caucasian hair had a higher change in volume compared to the Asian hair in variable and high relative humidity conditions. The hair volume increase when subject to high air humidity, and it was lower with the incorporation of a peptide into climate control formulations. CONCLUSION: Caucasian hair showed higher volume than Asian hair when submitted to both relative humidity conditions. The incorporation of the peptide into the climate control formulations, a base (mostly composed of water ≈ 94%) and an ethanolic, was found to reduce the volume of Caucasian hair tresses. The presence of the peptide improved the hair volume change more than 60% in high relative humidity conditions.

Assessment of penetration of Ascorbyl Tetraisopalmitate into biological membranes by molecular dynamics.

The present work, involves the simulation of the transport of a vitamin C derivative, Ascorbyl Tetraisopalmitate (ATI), through human skin by molecular dynamics. Percutaneous absorption of the ATI molecule through the infundibulum, an important route of absorption into the hair follicle of the human skin, has been modeled and compared with the stratum corneum membrane. The comparative study was done using molecular dynamics with Martini force field. In infundibulum, a single ATI molecule require more time to penetrate, and the data obtained suggested that a high concentration of ATI molecule accelerated the process of penetration. In conclusion, the ATI molecule was found to have more affinity towards the stratum corneum as compared with the infundibulum, and it followed a straight pathway to penetrate (until 600ns of simulation). In the infundibulum, it showed less affinity, more mobility and followed a lateral pathway. Thus, this work contributes to a better understanding of the different molecular interactions during percutaneous absorption of active molecules in these two different types of biological membranes.

Improved Poly (D,L-lactide) nanoparticles-based formulation for hair follicle targeting.

OBJECTIVE: Hair follicles are widely recognized as the preferential target and site of accumulation for nanoparticles after topical application. This feature is of particular importance for hair cosmetics, having the potential to refine the treatment of several hair follicle-related disorders. The aim of this work was to improve the preparation of Poly (D,L-lactide) (PLA) nanoparticles for in vivo follicular target and drug delivery. METHODS: Envisaging a future industrial scale-up of the process, nanoprecipitation method was used to prepare PLA nanoparticles: the effect of several processing parameters on their properties was examined and the yield of nanoparticles formation determined. Encapsulation efficiencies and in vitro release profiles of lipophilic and hydrophilic model compounds were also assessed. In vitro cytotoxicity and ex vivo penetration studies were performed on a reference skin cell line (NCTC2455, human skin keratinocytes) and porcine skin, respectively. RESULTS: Using acetone : ethanol (50 : 50, v/v) as the solvent phase, 0.6% (w/w) of Pluronic(®) F68 as a surfactant agent and agitation to mix the solvent and non-solvent phases, a monodispersed population of non-cytotoxic spherical nanoparticles of approximately 150 nm was obtained. The yield of nanoparticles for this formulation was roughly 90%. After encapsulation of model compounds, no significant changes were found in the properties of particles and the entrapment efficiencies were above 80%. The release kinetics of dyes from PLA nanoparticles indicate an anomalous transport mechanism (diffusion and polymer degradation) for Nile Red (lipophilic) and a Fickian diffusion of first order for fluorescein 5(6)-isothiocyanate (hydrophilic). Ex vivo skin penetration studies confirmed the presence of nanoparticles along the entire follicular ducts. CONCLUSIONS: The optimized method allows the preparation of ideal PLA nanoparticles-based formulations for hair follicle targeting. PLA nanoparticles can effectively transport and release lipophilic and hydrophilic compounds into the hair follicles, and the yields obtained are acceptable for industrial purposes.

Lipases efficiently stearate and cutinases acetylate the surface of arabinoxylan films.

This is the first report on successful enzyme catalyzed surface esterification of hemicellulose films. Enzyme catalyzed surface acetylation with vinyl acetate and stearation with vinyl stearate were studied on rye arabinoxylan (AX) films. Different surface analytical techniques (FT-IR, TOF-SIMS, ESCA, CA) show that lipases from Mucor javanicus, Rhizopus oryzae and Candida rugosa successfully surface stearate AX films and that a cutinase from Fusarium solani pisi surface acetylates these films. The specificities of cutinase and lipases were also compared, and higher activity was observed for lipases utilizing long alkyl chain substrates while higher activity was observed for cutinase utilizing shorter alkyl chain substrates. The contact angle analysis showed films with increased initial hydrophobicity on the surfaces.

Potential of human γD-crystallin for hair damage repair: insights into the mechanical properties and biocompatibility.

OBJECTIVES: The objective of this work was to develop a new strategy to physically 'repair' chemically damaged hair. Hence the human eye γD-crystallin, a protein from the superfamily characterized structurally by the Greek key motif, was studied. The human γD-crystallin was chosen based on the ability of proteins belonging to this superfamily to be involved in the coating of specific structures. Two crystallins were used on the study, the wild type (Protein Data Bank ID: 1HK0) and the mutant protein. The mutant form was intended to induce a strong and quick protein polymerization as well to have new possible points of anchorage to hair. METHODS: The ability of both crystallins to bind to damaged hair and even penetrate into its cortex was checked by fluorescence microscopy, confocal microscopy and scanning electron microscopy. Furthermore the reinforcement of hair mechanical resistance, the potential cytotoxic/inflammatory effect of crystallins were studied in order to have a fully comprehension about the protein based formulation. RESULTS: Although the chemical over-bleaching treatment induced a decrease of 20% on the resistance of the hair, the crystallins which bind and penetrate the hair fibre were able to recover and even to improve its mechanical properties when compared to the virgin hair. Moreover none of the crystallins displayed a toxic effect in fibroblasts for all the range of tested concentrations upon 72 h of exposure. The active aggregation process of mutant crystallin induced an inflammatory response in fibroblasts in the first 24 h of contact, measured by the amount of released pro-inflammatory cytokine IL-6 to the medium. In contrast contact with wild type crystallin did not lead to significant inflammation. CONCLUSION: Outcome from protein formulation characterization supports the hypothesis that the γD-crystallin it is able to recover and improve the mechanical properties of chemical damaged hair. Therefore it can be considered as a very promising strengthening agent for the development of new restorative hair care products.

In vitro induction of melanin synthesis and extrusion by tamoxifen.

OBJECTIVES: Physical appearance has significant importance psychologically and socially, with skin and hair being of prime relevance. Effective ingredients that modulate melanin synthesis are of growing interest. Tamoxifen, a widely used selective oestrogen receptor modulator, SERM, was described occasionally in medical case reports as causing grey hair repigmentation. This work aimed to study, in vitro, the effect of tamoxifen and 4-hydroxy-tamoxifen, one of its most bioactive derivatives, on melanin production in human melanocytes. METHODS: Adult normal human epidermal melanocytes (NHEM) were treated with physiological concentrations of tamoxifen and 4-hydroxy-tamoxifen during 72 hours. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) leakage. Total melanin was quantified by spectrophotometry, and cyclic adenosine monophosphate (cAMP) was determined by competitive ELISA. The relative mRNA levels of several genes involved in melanogenesis were investigated by real-time PCR. RESULTS: Under the conditions used, the results showed that tamoxifen and 4-hydroxy-tamoxifen treatments, none of them toxic to NHEM, induced a time-dependent increase in the amount of melanin released to the culture medium. cAMP, one of the major second messenger in signalling pathways important to melanogenesis, was decreased after treatment. The transcript levels of genes coding for catalase, premelanosome protein and melan-A, directly related to skin and hair pigmentation, showed an increased tendency upon tamoxifen and 4-hydroxy-tamoxifen treatment. Induction of catalase gene expression in NHEM points towards a promelanogenic effect mediated by ROS. CONCLUSION: According to the results, even in such a short treatment period, tamoxifen and 4-hydroxy-tamoxifen promoted melanin extrusion and they seem to act as melanogenesis stimulators at the molecular level. Our data suggest that SERMs might be a new tool for increasing melanogenesis and might be of great interest for topical formulations in cosmetic industry.

Keratins and lipids in ethnic hair.

Human hair has an important and undeniable relevance in society due to its important role in visual appearance and social communication. Hair is mainly composed of structural proteins, mainly keratin and keratin associated proteins and lipids. Herein, we report a comprehensive study of the content and distribution of the lipids among ethnic hair, African, Asian and Caucasian hair. More interestingly, we also report the study of the interaction between those two main components of hair, specifically, the influence of the hair internal lipids in the structure of the hair keratin. This was achieved by the use of a complete set of analytical tools, such as thin layer chromatography-flame ionization detector, X-ray analysis, molecular dynamics simulation and confocal microscopy. The experimental results indicated different amounts of lipids on ethnic hair compositions and higher percentage of hair internal lipids in African hair. In this type of hair, the axial diffraction of keratin was not observed in X-ray analysis, but after hair lipids removal, the keratin returned to its typical packing arrangement. In molecular dynamic simulation, lipids were shown to intercalate dimers of keratin, changing its structure. From those results, we assume that keratin structure may be influenced by higher concentration of lipids in African hair.

Keratin-based peptide: biological evaluation and strengthening properties on relaxed hair.

A peptide based on a fragment of hair keratin type II cuticular protein, keratin peptide (KP), was studied as a possible strengthening agent for weakened relaxed hair. The peptide was prepared both in aqueous water formulation (WF) and organic solvent formulations (OF), to determine the effect of organic solvents on peptide interaction with hair and the differences in hair recovery. Both peptide formulations were shown to improve mechanical and thermal properties of weakened hair with peptide in OF showing the stronger effect. As a potential new hair care product, and so would necessitate contact with skin, the cytotoxicity and genotoxicity of the peptide were also evaluated through different methodologies (Alamar Blue assay, 2'-7'-dichlorofluorescein probe, cell morphology and growth and evaluation of DNA damage by an alkaline version of the comet assay) in skin fibroblasts. These tests are indicators of the potential of peptide to cause irritation on skin or to be carcinogenic, respectively. The peptide in WF did not cause cytotoxicity or genotoxicity in any of the concentrations tested. The presence of OF, however, induced a 20% decrease in cell viability in all of the range of concentrations used after 72-h incubation. Moreover, OF inhibited cell growth and was considered genotoxic at first contact with cells. The peptide was therefore considered a promising strengthening agent for hair and was shown to be innocuous when applied in WF.

Liposome formation with wool lipid extracts rich in ceramides.

Internal wool lipids (IWLs) are rich in cholesterol, free fatty acids, cholesteryl sulfate, and, mainly, ceramides. The repairing effect of these lipids structured as liposomes was demonstrated by reinforcing the skin-barrier integrity and increasing the water-holding capacity when applied onto the skin. This work was focused on the formation of liposomes with IWLs rich in ceramides, obtained at pilot plant level with organic solvent extraction by using methanol and acetone. The lipid composition of the two extracts was quantitatively analyzed. IWL extracts containing different amounts of sterol sulfate were used to form liposomes at physiologic p(H). Vesicle size distribution, polydispersity index, and zeta potential of all liposomes were determined to characterize them and to study their stability. The results obtained showed that IWL extract composition, which was different depending on the extraction methodologies used, greatly influences the characteristics of the liposomes formed. Vesicular size and polydispersity index liposomes were smaller when the extract composition contained a higher proportion of either free fatty acids or sterol sulfate. Moreover, liposome stability was improved when some amount of sterol sulfate was added to the composition of methanol and acetone extracts. This natural mixture with keratinaceous origin could have a special interest for cosmetic or dermopharmaceutical companies.

Enzymatic hydrolysis of PTT polymers and oligomers.

Oligomers and polymers (film, fabrics) of the linear aromatic polyester poly(trimethylene terephthalate) (PTT) were treated with polyesterases from Thermomyces lanuginosus, Penicillium citrinum, Thermobifida fusca and Fusarium solani pisi. The cutinase from T. fusca was found to release the highest amounts of hydrolysis products from PTT materials and was able to open and hydrolyse a cyclic PTT dimer according to RP-HPLC-UV detection. In contrast, the lipase from T. lanuginosus also showed activity on the PTT fibres and on bis(3-hydroxypropyl) terephthalate (BHPT) but was not able to hydrolyse the polymer film, mono(3-hydroxypropyl) terephthalate (MHPT) nor the cyclic dimer of PTT. As control enzymes inhibited with mercury chloride were used. Surface hydrophilicity changes were investigated with contact angle measurements and the degree of crystallinity changes were determined with DSC.

Enzymatic reduction of azo and indigoid compounds.

A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40 degrees C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N approximately 1 approximately ,N approximately 1 approximately -dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H(2)O(2)) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy.

Restricting detergent protease action to surface of protein fibres by chemical modification.

Due to their excellent properties, such as thermostability, activity over a broad range of pH and efficient stain removal, proteases from Bacillus sp. are commonly used in the textile industry including industrial processes and laundry and represent one of the most important groups of enzymes. However, due to the action of proteases, severe damage on natural protein fibres such as silk and wool result after washing with detergents containing proteases. To include the benefits of proteases in a wool fibre friendly detergent formulation, the soluble polymer polyethylene glycol (PEG) was covalently attached to a protease from Bacillus licheniformis. In contrast to activation of PEG with cyanuric chloride (50%) activation with 1,1'-carbonyldiimidazole (CDI) lead to activity recovery above 90%. With these modified enzymes, hydrolytic attack on wool fibres could be successfully prevented up to 95% compared to the native enzymes. Colour difference (DeltaE) measured in the three dimensional colour space showed good stain removal properties for the modified enzymes. Furthermore, half-life of the modified enzymes in buffers and commercial detergents solutions was nearly twice as high as those of the non-modified enzymes with values of up to 63 min. Out of the different modified proteases especially the B. licheniformis protease with the 2.0-kDa polymer attached both retained stain removal properties and did not hydrolyse/damage wool fibres.

A new cuticle scale hydrolysing protease from Beauveria brongniartii.

From a screening for the production of new proteases specific for cuticle scales, Beauveria brongniartii was selected producing an alkaline Ca(++) dependent protease. The purified had a molecular weight of 27 kDa and a pI value of 8.0. Substrate specificities of model substrates (wool with partially removed cuticles treated with SDS) were analyzed by protein release, dissolved organic carbon (DOC) and nitrogen analysis. The C/N ratio of released material turned out to be a good parameter to determine the site of action of proteases on fibres. Compared to other enzymes, the fungal protease preferentially hydrolyzed cuticle scales and has thus a potential for anti-shrinking pre-treatment of wool fabrics.

New enzyme-based process direction to prevent wool shrinking without substantial tensile strength loss.

In this paper a new enzymatic process direction is described for obtaining machine washable wool with acceptable quality. In general, application of protease enzyme technology in wool processing results in considerable loss of tensile strength by diffusion of the enzyme into the interior of wool fibers. To overcome this disadvantage enzymatic activity has been more targeted to the outer surface of the scales by improving the susceptibility of the outer surface scale protein for proteolytic degradation. This has been realized by a pretreatment of wool with hydrogen peroxide at alkaline pH in the presence of high concentrations of salt.

Specificities of a chemically modified laccase from Trametes hirsuta on soluble and cellulose-bound substrates.

Laccases could prevent fabrics and garments from re-deposition of dyes during washing and finishing processes by degrading the solubilized dye. However, laccase action must be restricted to solubilized dye molecules thereby avoiding decolorization of fabrics. Chemical modification of enzymes can provide a powerful tool to change the adsorption behaviour of enzymes on water insoluble polymers. Polyethylene glycol (PEG) was covalently attached onto a laccase from Trametes hirsuta. Different molecular weights of the synthetic polymer were tested in terms of adsorption behaviour and retained laccase activity. Covalent attachment of PEG onto the laccase resulted in enhanced enzyme stability while with increasing molecular weight of attached PEG the substrate affinity for the laccase conjugate decreased. The activity of the modified laccases on fibre bound dye was drastically reduced decreasing the adsorption of the enzyme on various fabrics. Compared to the 5 kDa PEG laccase conjugate (K/S value 47.60) the K/S value decreased much more (47.96-46.35) after the treatment of dyed cotton fabrics with native laccase.

Azo reductase activity of intact saccharomyces cerevisiae cells is dependent on the Fre1p component of plasma membrane ferric reductase.

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae Deltafre1 and Deltafre1 Deltafre2 mutant strains, but not the Deltafre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae Deltafre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.

Characterization of azo reduction activity in a novel ascomycete yeast strain.

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.

An immobilised catalase peroxidase from the alkalothermophilic Bacillus SF for the treatment of textile-bleaching effluents.

A catalase peroxidase (CP) from the newly isolated Bacillus SF was used to treat textile-bleaching effluents. The enzyme was stable at high pH values and temperatures, but was more sensitive to deactivation by hydrogen peroxide than monofunctional catalases. Based on the Michaelis-Menten kinetics of the CP, a model was developed to describe its deactivation characteristics. The enzyme was immobilised on various alumina-based carrier materials with different shapes and the specific activity increased with the porosity of the carrier. The shape of the carrier had an important influence on the release of oxygen formed during the catalase reaction from the packed-bed reactor and Novalox saddles were found to be the most suitable shape. Bleaching effluent was treated in a horizontal packed-bed reactor containing 10 kg of the immobilised CP at a textile-finishing company. The treated liquid (500 l) was reused within the company for dyeing fabrics with various dyes, resulting in acceptable colour differences of below Delta E*=1.0 for all dyes.